WO2002079215A1 - Synthons pour la synthese d'une phase soluble d'oligonucleotides - Google Patents

Synthons pour la synthese d'une phase soluble d'oligonucleotides Download PDF

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WO2002079215A1
WO2002079215A1 PCT/US2002/008547 US0208547W WO02079215A1 WO 2002079215 A1 WO2002079215 A1 WO 2002079215A1 US 0208547 W US0208547 W US 0208547W WO 02079215 A1 WO02079215 A1 WO 02079215A1
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nucleoside
levulinyl
lipase
hydrolase
under conditions
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PCT/US2002/008547
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English (en)
Inventor
Yogesh S. Sanghvi
Vicente Gotor
Miguel Ferrero
Susana Fernandez
Javier Garcia
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Isis Pharmaceuticals, Inc.
Univesidad De Oviedo
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Priority to KR10-2003-7012688A priority Critical patent/KR20040014479A/ko
Priority to EP02715168A priority patent/EP1379536A4/fr
Publication of WO2002079215A1 publication Critical patent/WO2002079215A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides

Definitions

  • the present invention relates to methods for the preparation of 3'-O and 5'-O- levulinyl nucleosides from common precursors using an enzymatic approach. These methods are useful for the large-scale synthesis of oligonucleotides.
  • oligonucleotides and oligonucleotide analogs as "antisense” agents.
  • the oligonucleotides or oligonucleotide analogs complimentary to a specific, target, messenger RNA (mRNA) sequence are used.
  • Antisense methodology is often directed to the complementary hybridization of relatively short oligonucleotides and oligonucleotide analogs to single-stranded mRNA or single- stranded DNA such that the normal, essential functions of these intracellular nucleic acids are disrupted.
  • Hybridization is the sequence specific hydrogen bonding of oligonucleotides or oligonucleotide analogs to Watson-Crick base pairs of RNA or single-stranded DNA. Such base pairs are said to be complementary to one another.
  • Oligonucleotides and oligonucleotide analogs are now accepted as therapeutic agents holding great promise for therapeutics and diagnostics methods. But applications of oligonucleotides and oligonucleotide analogs as antisense agents for therapeutic purposes, diagnostic purposes, and research reagents often require that the oligonucleotides or oligonucleotide analogs be synthesized in large quantities.
  • nucleoside monomer building blocks bearing protecting groups on the 3'-O and or the 5'-O positions The protecting groups should be stable to coupling conditions and selectively cleaved without affecting other protecting groups in the molecule.
  • One such protecting group is the levulinyl group, -C(O)-(C ⁇ 2 ) 2 -C(O)- CH 3 .
  • the preparation of nucleosides bearing these protecting groups involves several tedious chemical protection/de-protection and/or purification steps.
  • nucleosides can be accomplished using a well-established method wherein nucleosides are selectively acylated at their hydroxyl sites by reacting the nucleosides with levulinic acid in the presence of DCC
  • Candida antarctica lipase B catalyzes acylation at the 5 '-hydroxyl group of nucleosides with high selectivity.
  • Pseudomonas cepacia lipase shows unusual regioselectivity towards the secondary alcohol at the 3 '-position of 2'- deoxynucleosides.
  • the present inventors have discovered methods and reagents that are useful in, for example, the large-scale synthesis of oligonucleotides.
  • the methods of the present invention help to minimize the number of steps required to yield desired results using an enzymatic approach.
  • the present invention provides methods and reagents for synthesizing both 3'-O-levulinyl nucleosides and 5 '-O-levulinyl nucleosides from a common precursor.
  • the regioselective deprotection of a 3',5'-di-O-levulinyl nucleoside yields either a 3'- ⁇ levulinyl nucleoside or a 5 '-O-levulinyl nucleoside, depending upon parameters disclosed herein.
  • either the 5 '-O-levulinyl nucleoside or the 3'- O-levulinyl nucleoside can be selectively produced by contacting the corresponding 3',5'-di- O-levulinyl nucleoside precursor with a particular hydrolase, such as a lipase.
  • the present invention provides a method of selectively preparing a nucleoside having a levulinyl at either the 3'-O or 5'-O position by contacting the nucleoside with an acylating agent for introducing the levulinyl group in the presence of a selected hydrolase (e.g. a lipase).
  • a selected hydrolase e.g. a lipase
  • methods for regioselectively deprotecting a 3',5'-di-O-levulinylnucleoside, said methods comprising selecting a hydrolase, e.g. a lipase, that is effective to direct a regioselective hydrolysis of one of the levulinyl positions, without causing an undesired level of hydrolysis on the other of the levulinyl positions, and contacting the 3', 5'-di-O-levulinyl nucleoside with the lipase for a time and under conditions effective to yield either a 3 '-O-levulinyl or a 5 '-O-levulinyl nucleoside.
  • a hydrolase e.g. a lipase
  • hydrolases that are amenable to the present invention include the lipases Candida antarctica lipase B (CAL-B), Candida antarctica lipase A (CAL-A), Pseudomonas cepacia lipase (PSL), porcine pancreatic lipase, Chromobacteriaum viscosum lipase, Mucor miehei lipase, Humicola lanuginosa lipase, Penicillium camemberti lipase, Candida rugosa lipase, and others.
  • CAL-B Candida antarctica lipase B
  • CAL-A Candida antarctica lipase A
  • Pseudomonas cepacia lipase PSL
  • porcine pancreatic lipase Chromobacteriaum viscosum lipase, Mucor miehei lipase, Humicola lanuginosa lipase, Penicillium camemberti lipa
  • methods for regioselectively acylating a nucleoside, said methods comprising selecting a hydrolase, such as a lipase, that is effective to direct a regioselective acylation of one of the -OH positions of the nucleoside, without causing an undesired level of acylation on the other of the levulinyl positions, and contacting the nucleoside with an acylating agent for introducing the levulinyl group and the selected lipase for a time and under conditions effective to yield either a 3 '-O-levulinyl or a 5'-O- levulinyl nucleoside.
  • a hydrolase such as a lipase
  • a 3',5'-di-O-levulinyl nucleoside is deprotected at the 5'- ⁇ -levulinyl position by contacting the diprotected nucleoside with CAL-B for a time and under conditions effective to regioselectively hydrolyze the 5 '-O-levulinyl position without affecting the 3 '-O-levulinyl position.
  • a nucleoside is selectively acylated at the 5'- ⁇ H position (or simply 5'- position) by contacting the nucleoside with an acylating agent for introducing the levulinyl group in the presence of CAL-B for a time and under conditions effective to regioselectively acylate the nucleoside at the 5 '-position to form the 5 '-O-levulinyl nucleoside.
  • a 3'-, 5'- di-O-levulinyl nucleoside is deprotected at the 3'-O levulinyl position by contacting the diprotected nucleoside with CAL-A or PSL-C for a time and under conditions effective to regioselectively hydrolyze the 3 '-O-levulinyl position without affecting the 5 '-O-levulinyl position.
  • a nucleoside is regioselectively acylated at the 3'-OH position by contacting the nucleoside with an acylating agent for introducing the levulinyl group in the presence of CAL-A or PSL-C for a time and under conditions effective to regioselectively acylate the nucleoside at the 3'-OH position to produce the 3 '-O-levulinyl nucleoside.
  • nucleoside has one of the following formulas:
  • R 1 is -H, -hydroxyl, a protected hydroxyl, a 2'-substituent or a 2' -protected substituent
  • R 2 and R 3 are, independently, -H or an amino protecting group
  • G is N or CH
  • Lev is -C(O)-(CH 2 ) 2 -C(O)-CH 3 , the levulinyl group; the methods comprising selecting a lipase that is effective to direct a regioselective hydrolysis of the 5 '-O-levulinyl position, without causing hydrolysis on the 3 '-O-levulinyl position, and contacting the 3', 5'-di-O-levulinyl nucleoside with the lipase for a time and under conditions effective to yield a 3 '-O-levulinyl nucleoside.
  • a preferred lipase for 5 '-O-levulinyl hydrolysis is CAL-B.
  • nucleoside has one of the following formulas:
  • R 6 is -H, -hydroxyl
  • R 2 , R , R 4 , and R 5 are each, independently, -H or an amino protecting group
  • G is N or CH
  • Lev is -C(O)-(CH 2 ) 2 -C(O)-CH 3 ; the methods comprising selecting a lipase that is effective to direct a regioselective hydrolysis of the 3 '-O-levulinyl position, without causing hydrolysis of the 5 '-O-levulinyl position, and contacting the 3', 5'-di-O-levulinyl nucleoside with the lipase for a time and under conditions effective to yield a 5 '-O-levulinyl nucleoside.
  • Lipases that are preferable for hydrolysis at the 3 '-O-levulinyl positions are, for example, CAL-A or PSL-C.
  • nucleoside has one of the following formulas:
  • Ri is -H, -hydroxyl, a protected hydroxyl, a 2'-substituent or a 2'-protected substituent
  • R 2 and R 3 are, independently, -H or an amino protecting group
  • G is N or CH; and Lev is -C(O)-(CH 2 ) 2 -C(O)-CH 3 , the levulinyl group; the methods comprising selecting a lipase that is effective to direct a regioselective acylation of the 5'-O position, without causing acylation of the 3'-O position, and contacting the nucleoside with an acylating agent for introducing the levulinyl group and the lipase for a time and under conditions effective to yield a 5 '-O-levulinyl nucleoside.
  • a preferred lipase for 5'- O-levulinyl acylation is CAL-B.
  • nucleoside has one of the following formulas:
  • R 6 is -H, -hydroxyl
  • R 2 , R 3 , R 4 , and R 5 are each, independently, -H or an amino protecting group
  • G is N or CH
  • Lev is -C(O)-(CH 2 ) 2 -C(O)-CH 3 ; the methods comprising selecting a lipase that is effective to direct a regioselective acylation of the 3'-O position, without causing acylation of the 5'-O position, and contacting the nucleoside with an acylating agent for introducing the levulinyl group and the lipase for a time and under conditions effective to yield a 3 '-O-levulinyl nucleoside.
  • Lipases that are preferable for regioselective acylation at the 3'- ⁇ position are, for example, CAL-A or PSL-C.
  • methods for acylating a hydroxyl moiety of a nucleic acid, such as a nucleoside or a nucleotide, at one or more of a 2'- O, 3'-O, or 5'-O position, said methods comprising reacting the nucleic acid with levulinic acid in the presence of a coupling agent, such as a carbodiimide, that is attached to a polymeric support for a time and under conditions effective to form an ester at the 2'-O, 3'-O or 5'-O position.
  • a coupling agent such as a carbodiimide
  • the present invention includes the esterification or acylation of any hydroxyl moiety, such as those found in carbohydrates or steroid molecules, by reacting the compounds containing the hydroxyl moiety with levulimc acid in the presence of a coupling agent that is attached to a polymeric support for a time and under conditions effective to form an ester between the hydroxyl moieties and the levulinyl group of the levulinic acid.
  • a coupling agent that is attached to a polymeric support for a time and under conditions effective to form an ester between the hydroxyl moieties and the levulinyl group of the levulinic acid.
  • B is a nucleobase
  • T ⁇ and T 2 are, independently, -hydroxyl, a hydroxyl protecting group, an activated phosphate group, a nucleotide, a nucleoside, or an oligonucleotide; R is -H, -hydroxyl, a protected hydroxyl or a 2' substituent group; provided that at least one of T ls T 2 or R is -hydroxyl; the methods comprising reacting said compound with levulinic acid in the presence of a coupling agent that is attached to a solid support, such as PS-cyclohexylcarbodiimide, for a time and under conditions effective to form an ester between the hydroxyl moiety and the levulinyl group.
  • T 1 and T 2 are -OH and R is -H or a 2'- substituent.
  • Bx is a nucleobase
  • R is hydroxyl or an optionally protected 2'-substituent comprising reacting said compound with levulinic acid in the presence of a coupling agent that is attached to a solid support for a time and under conditions effective to form a compound having formula:
  • Lev is -levulinyl
  • methods for generating a cyclohexylcarbodiimide derivatized polymeric support from a cyclohexylurea derivatized polymeric support, said methods comprising reacting the cyclohexylurea derivatized polymeric support with a dehydrating agent, such as tosyl chloride or POCl 3 , in an organic solvent for a time and under conditions effective to yield the cyclohexylcarbodiimide derivatized polymeric support.
  • the organic solvent employed is CH 2 C1 2 , CHC1 3 , hexane, or pyridine.
  • methods for generating a cyclohexylcarbodiimide derivatized polymeric support from a cyclohexylurea derivatized polymeric support, the methods comprising the steps of reacting the cyclohexylurea derivatized polymer support with a dehydrating agent for a time and under conditions effective to form a salt and subsequently contacting the salt with an aqueous solution, such as aqueous
  • Figure 1 shows 3', 5'-di-O-acylation bf a 2'-deoxynucleoside using levulinic acid and DCC or levulinic acid and PS-carbodiimide.
  • Figure 2 shows the enzymatic regioselective hydrolysis of a 3',5'-di-O-levulinyl 2'- deoxynucleoside.
  • Figure 3 shows the enzymatic regioselective hydrolyis of a 3',5'-di-O-levulinyl 2'- substituted nucleoside.
  • Figure 4 is a table depicting the results of regioselective hydrolysis of nucleosides 2a -
  • Figure 5 depicts the regioselective acylation of 2'-deoxynucleosides.
  • Figure 6 is a table depicting the results of regioselective acylation of 2'- deoxyribonucleosides la, lc, le and lg.
  • Figure 7 depicts the regioselective acylation of ribonucleosides.
  • Figure 8 is a table depicting the results of regioselective acylation of ribonucleosides 5a-5g.
  • the present invention is directed to the preparation of nucleoside building blocks such as 3', 5'-di-O-levulinylnucleosides, 3'-O-levulinylnucleosides, and 5'-O-levulinylnucleosides that are especially useful in the large-scale synthesis of oligonucleotides.
  • methods for protecting a hydroxyl moiety of a nucleic acid at at least one of a 2'-O, 3'-O or 5'-O position comprising reacting the nucleic acid with levulinic acid in the presence of a coupling agent that is attached to a polymeric support for a time and under conditions effective to form an ester at the 2'-O, 3'-O or 5'-O position.
  • the nucleic acids of the present invention include nucleosides, nucleotides, oligonucleosides and oligonucleotides.
  • the nucleic acid is a nucleoside and the polymeric support is a polystyrene support or a polyethylene glycol support that is coupled to a coupling agent, such as cyclohexylcarbodiimide.
  • the nucleic acid has the formula:
  • Bx is a nucleobase
  • Ti and T are, independently, hydroxyl, a protected hydroxyl, an activated phosphate group, a nucleotide, a nucleoside, or an oligonucleotide;
  • R is -H, -hydroxyl, a protected hydroxyl, or a 2' substituent group; provided that at least one of Ti, T 2 or R is -OH; the method comprising reacting said compound with levulinic acid in the presence of a coupling agent that is attached to a solid support for a time and under conditions effective to form an ester between the hydroxyl moiety and the levulinyl group.
  • T 1 and T 2 are -OH and R is H.
  • the protection methods of the present invention are not limited to acylation of the hydroxyl groups of nucleosides. Any hydroxyl functionality may be acylated using the methods of the present invention, including those found in carbohydrate or steroid molecules. According some embodiments of the present invention, referring to Figure 1, 3', 5'-di-
  • O-levulinyl nucleosides (2) were prepared from their corresponding natural nucleosides by treatment with levulinic acid and PS-carbodiimide in 1,4-dioxane in the presence of DMAP as a catalyst. Filtering off of the polystyrene beads removed the urea and the N-levulinylurea derivatives, which were polymer bound. 3', 5'-di-O-levulinylthymidine (2a) and 3',5'-di-O- levulinyl-2'-deoxyadenosine (2d) were isolated with 91% and 95% yield, respectively.
  • the PS-dicarbodiimide an expensive reagent, is recovered by reacting the cyclohexylurea derivatized polymer support with a dehydrating agent in an organic solvent.
  • a dehydrating agent include POCl 3 and tosyl chloride.
  • Preferred organic solvents include
  • 3', 5'-di-O-levulinyl nucleosides can alternatively be prepared from the corresponding natural nucleosides (1) by treatment with 5.2 equivalents of levulinic acid (LevOH) and dicyclohexylcarbodiimide (DCC) in 1,4-dioxane in the presence of DMAP as catalyst.
  • the reaction takes place through activation of the levulinic acid with
  • Regioselective deprotection of the common precursor, 3', 5'-di-O-levulinyl nucleoside, at the 5'-O-levulinyl position is effected by selecting a hydrolase, e.g. a lipase, effective to direct regioselective hydrolysis at the 5'-O -levulinyl position, without causing hydrolysis at the 3 '-O-levulinyl position, and contacting the diprotected nucleoside with the lipase for a time and under conditions effective to hydrolyze the 3', 5'-di- ⁇ -levulinyl nucleoside at the 5'- O-levulinyl position.
  • the diprotected nucleosides have one of the following formulas:
  • Ri is -H, -hydroxyl, a protected hydroxyl, or a 2'-substituent
  • R 2 and R 3 are, independently, -H or an amino protecting group
  • G is N or CH
  • Lev is -C(O)-(CH 2 ) 2 -C(O)-CH 3 .
  • TLC showed total disappearance of the starting material after 62h (entry 1, Table 1).
  • Table 1 shown in Figure 4, also indicates that substrates 3',5'-di-O-levulinyl cytosine
  • nucleosides are also successfully selectively deprotected at the 5'-O- levulinyl position.
  • all four nucleosides 2'-methoxy-3', 5'-di-O- levulinyladenosine (6a), 2'-methoxyethoxy-3', 5'-di-O-levulinyladenosine (6b), 2'methoxy- 3',5'-di-O-levulinyl-2'-deoxycytosine (6c), and 2'-methoxyethoxy-3', 5'-di-O-levulinyl-5- methyl cytosine (6d) were selectively hydrolyzed with CAL-B furnishing 7a-7d in high yields.
  • 3', 5'-di-O-levulinyl nucleosides are regioselectively deprotected at the 3 '-O-levulinyl position by selecting a hydrolase, e.g.
  • the diprotected nucleosides have one of the following formulas:
  • R 6 is -H, or -OH
  • R 2 , R 3 , R 4 , and R 5 are each, independently, -H or an amino protecting group
  • G is N or CH
  • Lev is -C(O)-(CH 2 ) 2 -C(O)-CH 3 .
  • 3'-O- selective hydrolysis was accomplished by reaction of 2 with immobilized Pseudomonas cepacia lipase [PSL-C, ratio of 1:3 w/w (2/PSL- C)] at 60 °C in 0.15M phosphate buffer giving the 5 '-O-levulinyl derivative.
  • PSL-C immobilized Pseudomonas cepacia lipase
  • CAL-A also exhibited excellent selectivity towards the 3 '-O-levulinyl position and has the advantage of requiring lower reaction temperatures than PSL-C (40 °C instead of 60 °C), shorter reaction times, and a lower ratio of enzyme/starting material (see Figure 4).
  • N-Benzoyl-di-levulinyl derivatives (2c) and (2e) were both appropriate substrates for both lipases, PSL-C and CAL-A.
  • enzymatic acylation may be carried out as depicted in Figure 5.
  • a suspension of 1 (0.2 mmol), the oxime ester (0.6 mmol), and the lipase in dry THF (1 mL) under nitrogen was stirred at 250 rpm for the time and at the temperature indicated in Table 3 ( Figure 8).
  • the reactions were monitored by TLC (10% MeOH/CH Cl 2 ).
  • the enzyme was filtered off and washed with CH 2 C1 2 , the solvents were distilled under vacuum, and the residue was taken up in ⁇ aHCO 3 (aq) and extracted with CH 2 C1 2 .
  • the combined organic layers were dried over Na 2 SO 4 and evaporated.
  • the residue was precipitated in diethyl ether to afford after filtration the monolevulinyl nucleosides 3 or 4. No further purification was necessary except in entries 10 and 11 to separate the traces of other acyl derivatives.
  • the acylation method requires fewer steps than the hydrolysis process, and is concomitantly more facile.
  • the starting materials are the parent nucleosides, and is not necessary to first make the dilevulinyl derivatives.
  • the hydrolysis method requires two steps, the acylation method requires only one.
  • the oxime ester is prepared in a single step and is purified by simple filtration. Flash chromatography can be avoided in the acylation process.
  • the acylation method may be applied to ribonucleosides, such as those set forth in formulae XNI-XX.
  • ribonucleosides such as those set forth in formulae XNI-XX.
  • a general acylation method for acylating ribonucleosides is depicted in Figure 7, while data pertaining thereto are set forth in
  • 3'-O-Lev-2'-O-MOE-5-Me-C Bz , 3'-O-Lev-2'-O-MOE-A Bz , and 3'-O-Lev- 2'-O-Me-A Bz were obtained using PSL-C. Hydrolysis reactions are required to prepare the corresponding 3 ' -Lev-T and 3 ' -Lev-G lBu derivatives.
  • nucleic acids useful in practicing the present invention include naturally occurring and non-naturally occurring nucleosides and nucleotides.
  • the nucleosides and nucleotides of the present invention are not limited to monomer units but may also contain a plurality of linked monomer units, to form dinucleosides, nucleotides, and oligonucleotides and comprise naturally and non-naturally occurring nucleobases, sugars, and backbones.
  • ⁇ on-naturally occurring nucleosides and nucleotides may be modified by replacing the sugar moiety with an alternative structure which has primary and secondary alcohol groups similar to those of ribose.
  • ⁇ on-naturally occurring sugars and nucleosidic bases are typically structurally distinguishable from, yet functionally interchangeable with, naturally occurring sugars (e.g. ribose and deoxyribose) and nucleosidic bases (e.g., adenine, guanine, cytosine, thymine).
  • non-naturally occurring nucleobases and sugars include all such structures which mimic the structure and/or function of naturally occurring species, and which aid in the binding of the oligonucleotide to a target, or which otherwise advantageously contribute to the properties of the oligonucleotide.
  • Backbone modifications include modifications to the phosphate backbone to increase the resistance to nucleases. These modifications include use of linkages such as methyl phosphonates, phosphorothioates and phosphorodithioates as well as those modifications that dramatically alter the nature of the internucleotide linkage such as non-phosphorus linkages, peptide nucleic acids (PNAs) and 2'-5' linkages.
  • a heterocyclic base moiety (often referred to in the art simply as a "base” or a
  • nucleobase amenable to the present invention includes both naturally and non-naturally occurring nucleobases.
  • the heterocyclic base moiety further may be protected wherein one or more functionalities of the base bears a protecting group.
  • unmodified or
  • nucleobases include the purine bases adenine and guanine, and the pyrimidine bases thymine, cytosine and uracil.
  • Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-pro ⁇ yl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl
  • nucleobases include those disclosed in United States Patent No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J.I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al, Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B., ed., CRC Press, 1993.
  • the processes of the present invention are applicable to so-called C-nucleosides (i.e. nucleosides in which the nucleobase is linked to the nucleoside ring via a C-C bond, as opposed to the naturally-occurring C-N bond).
  • the nucleoside ring is in the ⁇ - configuration (as opposed to the naturally-occurring ⁇ -configuration), the L-configuration (as opposed to the naturally-occurring D-configuration) or both (i.e. the ⁇ -L-configuration).
  • Certain heterocyclic base moieties are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention to complementary targets. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 °C (Id., pages 276-278) and are presently preferred base substitutions, even more particularly when combined with selected 2'-sugar modifications such as 2'-methoxyethyl groups.
  • a representative list of 2'-substituent groups amenable to the present invention include C ! -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl, C 5 -C 2 o aryl, O-alkyl, O-alkenyl, O-alkynyl, O- alkylamino, O-alkylalkoxy, O-alkylaminoalkyl, O-alkyl imidazole, S-alkenyl, S-alkynyl, NH- alkyl, NH-alkenyl, NH-alkynyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH- aralkyl, N-phthalimido, halogen (particularly fluoro), keto, carboxyl, nitro, nitroso, nitrile, trifiuoromethyl, tri
  • polyethers linear and cyclic polyethylene glycols (PEGs), and (PEG)-containing groups, such as crown ethers and those which are disclosed by Ouchi et al. (Drug Design and Discovery 1992, 9, 93), Ravasio et al (J. Org. Chem. 1991, 56, 4329) and Delgardo et. al. (Critical Reviews in Therapeutic Drug Carrier Systems 1992, 9, 249), each of which is herein incorporated by reference in its entirety. Further sugar modifications are disclosed in Cook, P.D., Anti-Cancer Drug Design, 1991, 6, 585-607.
  • Additional substituent groups amenable to the present invention include -SR and -NR 2 groups, wherein each R is, independently, hydrogen, a protecting group or substituted or unsubstituted alkyl, alkenyl, or alkynyl.
  • 2'-SR nucleosides are disclosed in United States Patent No. 5,670,633, issued September 23, 1997, hereby incorporated by reference in its entirety. The incorporation of 2'-SR monomer synthons are disclosed by Hamm et al, J. Org. Chem., 1997, 62, 3415-3420.
  • 2'-NR 2 nucleosides are disclosed by Goettingen, M., J. Org. Chem., 1996, 61, 73-6281; and Polushin et al, Tetrahedron Lett, 1996, 37, 3227-3230.
  • Further substiruent groups have one of formulae XXI or XXII:
  • Z 0 is O, S or NH
  • each R 6 , R 7 , R 8 , R 9 and R 10 is, independently, hydrogen, C(O)R , substituted or unsubstituted Ci-Cio alkyl, substituted or unsubstituted C 2 -C 10 alkenyl, substituted or unsubstituted C 2 -C 10 alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or a conjugate group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl; or optionally, R 7 and R 8 , together form a phthalimido moiety with the nitrogen atom to which they are attached; or optionally, R 9 and R 10 , together form a phthalimido moiety with the nitrogen atom to which
  • R 5 is T-L, T is a bond or a linking moiety
  • each Ri and R 2 is, independently, H, a nitrogen protecting group, substituted or unsubstituted -Cio alkyl, substituted or unsubstituted C 2 -C 10 alkenyl, substituted or unsubstituted C -C 10 alkynyl, wherein said substitution is OR 3 , SR 3 , NH 3 + , N(R 3 )(R t ), guanidino or acyl where said acyl is an acid amide or an ester; or Ri and R 2 , together, are a nitrogen protecting group or are joined in a ring structure that optionally includes an additional heteroatom selected from N and O; or Ri, T and L, together, are a chemical functional group; each R 3 and R* is, independently, H, - o alkyl, a nitrogen protecting group, or R 3 and R t , together, are a nitrogen protecting group; or R 3 and R are
  • R 5 is H or C C 8 alkyl
  • Z ⁇ , Z and Z 3 comprise a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero atoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is ahphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic;
  • Z 5 is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about
  • each c_ ⁇ is, independently, an integer from 1 to 10; each q 2 is, independently, 0 or 1; q 3 is 0 or an integer from 1 to 10; q 4 is an integer from 1 to 10; provided that when q 3 is 0, q 4 is greater than 1.
  • Representative substituent groups of Formula I are disclosed in United States Patent
  • Oligonucleotides hereby incorporated by reference in its entirety.
  • substituent groups include O[(CH 2 ) n O] m CH 3 , O(CH 2 ) n OCH 3 ,
  • O(CH 2 ) TakeNH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 (where n and m are from 1 to about 10), Ci to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3; OCF 3 , SOCH 3 , SO 2 CH 3> ONO 2; NO 2 , N 3, NH 2; heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino and substituted silyl.
  • Another particularly preferred modification includes 2'-methoxyethoxy (2'-O-CH 2 CH 2 OCH 3 or 2'-MOE, Martin et al, Helv. Chim. Ada, 1995, 78, 486).
  • a further preferred substituent group is 2'-dimethylaminooxyethoxy, i.e., a O(CH 2 ) ON(CH 3 ) group, also known as 2'- DMAOE.
  • nucleosides and oligomers include 2'-methoxy (2'-O-CH 3 ), 2'-aminopropoxy (2'- OCH 2 CH 2 CH 2 H 2 ) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on nucleosides and oligomers, particularly the 3' position of the sugar on the 3' terminal nucleoside or at a 3'-position of a nucleoside that has a linkage from the 2'-position such as a 2' -5' linked oligomer and at the 5'-position at a 5'-terminus. Oligomers may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • the methods of the present invention use labile protecting groups to protect various functional moieties during synthesis.
  • Protecting groups are used ubiquitously in standard oligonucleotide synthetic regimes for protection of several different types of functionality. In general, protecting groups render chemical functionality inert to specific reaction conditions and can be appended to and removed from such functionality in a molecule without substantially damaging the remainder of the molecule. See, e.g., Green and Wuts, Protective Groups in Organic Synthesis, 2d edition, John Wiley & Sons, New York, 1991.
  • Representative protecting groups useful to protect nucleotides during synthesis include base labile protecting groups and acid labile protecting groups. Base labile protecting groups are used to protect the exocyclic amino groups of the heterocyclic nucleobases.
  • This type of protection is generally achieved by acylation.
  • Two commonly used acylating groups for this purpose are benzoyl chloride and isobutyryl chloride. These protecting groups are stable to the reaction conditions used during oligonucleotide synthesis and are cleaved at approximately equal rates during the base treatment at the end of synthesis.
  • Hydroxyl protecting groups typically used in oligonucleotide synthesis may be represented by the group having the formula: -C(R (R 2 )(R 3 ) wherein each of R ls R 2 and R 3 is an unsubstituted or mono-substituted aryl or heteroaryl group selected from phenyl, naphthyl, anthracyl, and five or six membered heterocylic rings with a single heteroatom selected from N, O and S, or two N heteroatoms, including quinolyl, furyl, and thienyl; where the substituent is selected from halo (i.e., F, Cl, Br, and I), nitro, C ⁇ -C -aJJkyl or alkoxy, and aryl, aralkyl and cycloalkyl containing up to 10 carbon atoms; and wherein R 2 and R 3 may each also be CrC - alkyl or aralkyl or cycloalkyl containing up
  • an acylating agent for introducing the levulinyl group is an acylating agent that is capable of reacting with an active group, such as hydroxyl, to produce an acylated group, wherein the acyl portion is the levulinyl group as defined above.
  • Such acylating reagents include levulinic acid, as well as activated forms of levulinic acid, including the anhydride, esters, oximes and halic acid derivatives thereof.
  • Levulimc acid is a preferred acylating agent for introducing the levulinyl group.
  • the present invention contemplates the use of a hydrolase, such as a lipase, to affect either deprotection or acylation of a nucleoside or modified nucleoside.
  • a hydrolase such as a lipase
  • the present invention contemplates the use of such hydrolases in the acylation (to introduce a levulinyl group onto a nucleoside) or deprotection (i.e.
  • hydrolase thus includes lipases, proteases, and similar hydrolases capable of regioselectively directing acylation of a nucleoside or deprotection of an acylated nucleoside.
  • Candida antarctica lipase B (CAL-B) was a gift from Novo Nordisk Co.
  • Candida antarctica lipase A (CAL-A) and immobilized Pseudomonas cepacia lipase (PSL-C) were purchased from Roche Diagnostics S. L. and Amano Pharmaceuticals, respectively.
  • PS- carbodiimide was purchased from Argonaut Technologies (San Carlos, CA, EE.UU.). All other reagents were purchased from Aldrich or Fluka. Solvents were distilled over an adequate desiccant under nitrogen.
  • Method B To a stirred mixture of 1 (0.4 mmol) and Et 3 N (0.15 mL, 1 mmol) in 1,4- dioxane (5 mL) under nitrogen, was added levulinic acid (0.14 g, 1.2 mmol), PS-carbodiimide (1.05 g, 1.2 mmol), DMAP (4 mg, 0.032 mmol), and DMAP ⁇ C1 (3 mg, 0.02 mmol). The reaction was stirred at room temperature for 3 hours. The insoluble material was collected by filtration and the filtrate was evaporated under vacuum. The residue was taken up in NaHCO 3 (aq) and extracted with CH 2 C1 2 . The combined organic extracts were dried over Na 2 SO and evaporated. The solid was washed with cold Et 2 ⁇ to afford 3',5'-di-O-levulinylnucleosides 2a and 2d.
  • the present invention provides facile methodologies for selectively preparing 3 '-O-levulinyl nucleosides and 5 '-O-levulinyl nucleosides from nucleoside and/or 3', 5'-di- ⁇ -levulinyl nucleoside precursors.
  • the present invention is adaptable to large-scale preparation of protected nucleosides.
  • Compounds prepared by the inventive methods are useful in a variety methods for preparation of oligonucleotides.

Abstract

La présente invention concerne des méthodes de préparation de nucléosides de lévulinyle 3'-0 et 5'-0 à partir de précurseurs communs par utilisation d'une approche enzymatique.
PCT/US2002/008547 2001-03-30 2002-03-20 Synthons pour la synthese d'une phase soluble d'oligonucleotides WO2002079215A1 (fr)

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EP02715168A EP1379536A4 (fr) 2001-03-30 2002-03-20 Synthons pour la synthese d'une phase soluble d'oligonucleotides

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EP1710249A1 (fr) * 2004-01-27 2006-10-11 Nippon Shinyaku Co., Ltd. Compose d'acide ribonucleique et procede de synthese en phase liquide de compose d'acide oligonucleique
WO2014017615A1 (fr) * 2012-07-25 2014-01-30 国立大学法人高知大学 Monomère destiné à la synthèse d'arn, son procédé de préparation et procédé de préparation d'arn
US11725073B2 (en) 2020-12-29 2023-08-15 Hongene Biotech Corporation Compositions and methods for liquid phase oligonucleotide synthesis
US11851454B2 (en) 2021-12-30 2023-12-26 Hongene Biotech Corporation Compositions and methods for liquid phase oligonucleotide synthesis

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US6677120B2 (en) * 2001-03-30 2004-01-13 Isis Pharmaceuticals, Inc. Building blocks for the solution phase synthesis of oligonucleotides
US20040058886A1 (en) * 2002-08-08 2004-03-25 Dharmacon, Inc. Short interfering RNAs having a hairpin structure containing a non-nucleotide loop
CN101573370B (zh) 2006-10-10 2013-09-11 美迪维尔公司 Hcv核苷类抑制剂
CN105701026A (zh) * 2016-01-04 2016-06-22 上海斐讯数据通信技术有限公司 一种数据采集器及其利用系统冗余资源采集数据的方法

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Publication number Priority date Publication date Assignee Title
EP1710249A1 (fr) * 2004-01-27 2006-10-11 Nippon Shinyaku Co., Ltd. Compose d'acide ribonucleique et procede de synthese en phase liquide de compose d'acide oligonucleique
EP1710249A4 (fr) * 2004-01-27 2008-02-20 Nippon Shinyaku Co Ltd Compose d'acide ribonucleique et procede de synthese en phase liquide de compose d'acide oligonucleique
WO2014017615A1 (fr) * 2012-07-25 2014-01-30 国立大学法人高知大学 Monomère destiné à la synthèse d'arn, son procédé de préparation et procédé de préparation d'arn
US9303056B2 (en) 2012-07-25 2016-04-05 Kochi University Monomer for synthesis of RNA, method for producing same, and method for producing RNA
US11725073B2 (en) 2020-12-29 2023-08-15 Hongene Biotech Corporation Compositions and methods for liquid phase oligonucleotide synthesis
US11851454B2 (en) 2021-12-30 2023-12-26 Hongene Biotech Corporation Compositions and methods for liquid phase oligonucleotide synthesis

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US20020142307A1 (en) 2002-10-03
KR20040014479A (ko) 2004-02-14
EP1379536A1 (fr) 2004-01-14
US6677120B2 (en) 2004-01-13

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